Objectives: To establish the highest practical dose and frequency (HPDF) of 0.3% or 1% tenofovir vaginal gel applied once or twice daily by sexually abstinent HIV-uninfected women, and to evaluate the safety, tolerability and systemic pharmacokinetics of the HPDF in abstinent and sexually active HIV-negative and HIV-infected women.
Methods: Eighty-four women, enrolled in sequential cohorts, used the study product for 14 consecutive intermenstrual days. Safety laboratory assessments and pelvic examinations were carried out during five study visits, with colposcopy at enrollment and on day 14. Samples for pharmacokinetics were collected before and after the initial tenofovir gel use and at day 13.
Results: The 1% tenofovir gel used twice daily was as well tolerated as other regimens used by the 48 HIV-negative sexually abstinent women, establishing the HPDF. Although 92% of the women reported at least one adverse event, the majority were mild (87%) and involved the genitourinary tract (70%). One possibly product-related severe adverse event involving lower abdominal cramping was reported by a sexually abstinent woman who used 0.3% gel twice daily. Serum tenofovir levels were low but detectable in 14 of the 25 women. No new HIV RNA resistance mutations were detected after 2 weeks of tenofovir gel in the 24 HIV-infected participants. No significant systemic toxicity was detected.
Conclusion: A 2-week course of 1% tenofovir vaginal gel used twice daily was well tolerated in sexually abstinent and sexually active HIV-negative and HIV-positive women. Systemic tenofovir absorption occurred. Expanded safety and effectiveness testing is warranted.
From the aMiriam Hospital/Brown University, Providence, Rhode Island and Fenway Community Health, Boston, Massachusetts
bUniversity of Pennsylvania, Philadelphia, Pennsylvania
cFred Hutchinson Cancer Research Center, Seattle, Washington State
dHarlem Hospital Center and Columbia University, New York
eBronx-Lebanon Hospital Center, New York
fFamily Health International, Arlington, Virginia
gJohns Hopkins University, Baltimore, Maryland
hGilead Sciences Inc., Foster City, California
iNational Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.
Received 18 July, 2005
Revised 13 September, 2005
Accepted 3 October, 2005
Correspondence to Dr Kenneth H. Mayer, The Miriam Hospital, 164 Summit Avenue, Providence, Rhode Island 02906, USA. E-mail: Kenneth_Mayer@brown.edu
With almost 5 million new HIV infections anticipated to be transmitted through sexual intercourse in the next year, more effective preventive technologies are urgently needed . Although male condoms are protective , women often cannot negotiate their effective use [3–5]. The female condom is relatively costly and requires skill and cultural acceptance . A preventive HIV vaccine remains elusive . Acceptable, affordable, female-controlled prevention methods could be helpful in halting the epidemic [5,7–11]. Microbicides are topically applied products designed to prevent the sexual transmission of HIV and other infections [3–5,12]. Nonoxynol-9, a commercially available surfactant spermicide, showed activity against HIV and sexually transmitted infections in vitro and was evaluated as a topical microbicide . However, clinical trials suggested that mucosal ulcerations and inflammation caused by nonoxynol-9 could increase the ease of HIV transmission [13–15]. Consequently, increasing attention has been given to developing microbicides with different mechanisms of action, and with better safety and tolerability profiles.
Tenofovir gel, 9-[(R)-9-(2-phosphonylmethoxyprophyl)propyl]adenine monohydrate, a nucleotide reverse transcriptase inhibitor, has demonstrated ability to inhibit retroviral replication in animals and humans, and it has been well tolerated when used orally, as tenofovir disoproxil fumarate, (tenofovir DF; Viread) [16–20]. Tenofovir DF has been approved for treatment of HIV-1 infection and is increasingly used as part of therapeutic regimens for HIV-positive individuals . Tenofovir has been proven to be effective in blocking the transmission of SIV in animal models when given as pre- or postexposure prophylaxis systemically or when applied as an intravaginal gel [22–25]. Tenofovir bisphosphate, the active intracellular moiety, has a very long intracellular half-life (> 72 h), which could allow for more convenient, coitally independent intravaginal use . Given the data showing animal protection with tenofovir gel, and the extensive human safety data with oral tenofovir in HIV-positive patients, the HIV Prevention Trials Network (HPTN) decided to assess the safety and tolerability of tenofovir gel in HIV-negative and HIV-positive women and their male sexual partners (HPTN 050).
This trial was a multisite open-label phase I, stepped parallel dose-ranging and frequency study of tenofovir vaginal gel conducted among 84 women and 24 male sexual partners recruited by the Harlem Hospital Center and Bronx-Lebanon Hospital/Center in New York; the University of Pennsylvania, Philadelphia; and the Miriam Hospital, Providence, Rhode Island. The primary objectives were to assess the safety and toxicity of tenofovir gel on genital mucosa, and the systemic safety and toxicity of tenofovir gel in escalating doses and frequencies in low-risk HIV-negative women and then in HIV-positive women. The study also assessed acceptability and adherence, changes in vaginal flora and cervicovaginal HIV detection, and HIV resistance.
Each cohort enrolled 12 women, and if not more than one serious adverse event was detected among the women in that cohort, recruitment of the next cohort was able to proceed. The protocol called for a review of the safety and tolerability data once the first four cohorts had completed 2 weeks of gel use and follow-up, to determine the highest practical dose and frequency. Subsequently, HIV-negative, sexually active women and their male partners were recruited, followed by HIV-positive sexually abstinent women, followed by sexually active HIV-positive women and their male partners.
Participants were recruited through community educational activities, local newspaper advertisements, by flyers on college campuses, clinics and other locales, and word of mouth. HIV-positive women were recruited via flyers and videos in HIV care clinics, community-based organizations, and by word of mouth.
Informed consent and eligibility criteria
All participants underwent a consent process approved by local institutional review boards. Entry criteria for females included being 18–45 years of age, having regular menstrual cycles or being amenorrheic owing to long-acting progestin, and agreeing to all the protocol-specified procedures. Low-risk, abstinent women were defined as having no sexually transmitted infections in the prior 6 months, no sexual intercourse for the prior 3 months, and no history of injection drug use. Sexually active HIV-negative women met the same inclusion criteria except that they had to be in a mutually monogamous relationship with a low-risk, HIV-negative male partner for at least 3 months and had to agree to have protected sexual intercourse at least twice a week during 14 days of product use. Couples had to agree not to engage in oral or anal sex, or to use non-study condoms, lubricants, sex toys, spermicides or female-controlled barrier protection during the 14 days when study product was used. HIV-negative women and men underwent HIV antibody testing to confirm their status. HIV-positive women and men had to have documentation of prior HIV infection, CD4 lymphocyte counts ≥ 200 cells/μl at the screening visit, HIV plasma RNA ≤ 10 000 copies/ml if taking antiretroviral therapy or plasma HIV RNA < 55 000 copies/ml for at least the past 3 months if treatment naive. Inclusion criteria for HIV-uninfected male partners included being at least 18 years of age, in a mutually monogamous relationship with the study participant for at least 3 months, and having a negative HIV test. HIV-positive male partners had to have documentation of their HIV status and be in a stable, monogamous relationship with the participant for at least 3 months.
A screening visit to determine eligibility was scheduled for all potential study participants. During this visit, a medical history and behavioral assessment were completed. A physical examination, including a pelvic examination, was conducted for all participants, and Papanicalou smears were taken if confirmation of a normal test in the prior 6 months was not available. Blood was drawn for complete blood count, kidney and liver function tests, syphilis serology, as well as for HIV antibody testing. Samples for HIV plasma RNA and CD4 cell count measurements were drawn for HIV-positive participants. Urine was tested for gonorrhea and chlamydia by ligase chain reaction (Abbott Laboratories, North Chicago, Illinois, USA) until it was commercially unavailable; then the ProbeTec strand amplification method (Becton-Dickinson, Franklin Lakes, New Jersey, USA) was used. Urine pregnancy tests were performed. Male partners were clinically evaluated prior to enrollment.
Female participants underwent a pelvic examination, including colposcopy and digital imaging photography at enrollment. HIV-positive participants also had cervicovaginal lavage and Sno-strips (wicking paper) collected to detect genital HIV. Participants were given supplies of study product and instructed on its application and use. Tenofovir gel is a transparent viscous gel, with each tube of study product containing 6 g 0.3% or 1% tenofovir gel formulated in purified water with edetate disodium, citric acid, glycerin, methylparaben, propylparaben, hydroxyethylcellulose, and pH adjusted to 4.5. The polyethylene applicator was capable of administering a 4 g dose of gel (12 or 40 mg tenofovir).
Participants assigned to once daily dosing applied the gel at bedtime. Participants assigned to twice daily dosing applied the gel in the morning and at bedtime. Participants in the sexually active cohorts substituted a dose not more than 2 h before vaginal sexual intercourse for either the morning or bedtime dose on at least 2 days per week. Six participants from cohorts A2, B, C, and D in the pharmacokinetic cohorts had blood drawn prior to inserting the first dose of tenofovir gel and then 30 min after inserting the gel and 1, 2, 4, 6, 8, 12 and 24 h later. They returned to the clinic on day 13 to have their blood drawn prior to inserting tenofovir gel. Study staff evaluated participants 48–72 h after initiation of product use to inspect the cervicovaginal mucosa and to assess participants' tolerance and adherence. Participants returned for a day 7 visit, which included a pelvic examination (with cervicovaginal lavage and Sno-strip collection for HIV-positive participants) and a safety laboratory assessment. After 14 days of product use, participants returned and were questioned about their experiences using the tenofovir gel. Pelvic examinations with colposcopy and digital imaging were performed. Seven days after the HIV-negative women stopped using tenofovir gel, the study nurse contacted them. If they reported any new or recurring adverse events, they were asked to return for a follow-up visit. Seven days after the HIV-positive women stopped using tenofovir gel, they returned for a follow-up visit, which included a safety laboratory assessment, and a pelvic examination with the collection of cervicovaginal lavage and Sno-strips.
HIV-1 RNA quantification
Plasma HIV RNA levels were determined by the Roche Amplicor Monitor Ultrasensitive Assay, version 1.5 (Roche Diagnostics, Branchburg, New Jersey, USA). For viral load analysis of cervicovaginal lavage samples, HIV-1 RNA was extracted from 0.5 ml cervicovaginal lavage using the Boom method . The viral loads of the extracted RNA samples were determined using the Roche Amplicor assay.
Plasma and cervicovaginal lavage samples with viral loads > 500 copies/ml were analyzed using the ViroSeq HIV-1 Genotyping System, v2.5 (Celera Diagnostics, Alameda, California, USA). Plasma samples were analyzed according to the manufacturer's instructions. Cervicovaginal lavage samples were analyzed using the same methods, except that 1.0 ml lavage fluid was used for analysis. Electropherograms generated with the ViroSeq system were inspected for the presence of amino acid substitutions at positions in HIV-1 reverse transcriptase that are associated with resistance or hypersusceptibility to tenofovir (http://hivdb.stanford.edu/cgi-bin/NRTIResiNote.cgi).
Tenofovir concentration was assayed in human serum using a validated high-performance liquid chromatography and mass spectrometry detection method (MDS Pharma Services, Montreal, Canada). The assay had a dynamic range of 3.0–602.8 ng/ml with intra-run quality control sample precision and accuracy of 3.1–6.8% and 98.2–106.6%, respectively.
Adherence and acceptability assessments
Throughout the study, participants recorded information in study-provided diaries regarding their sexual activity, product and condom use, and symptoms. Information on factors influencing adherence to the study protocol was collected via an interviewer-administered questionnaire. Non-adherence was defined as 3 or more days of missed product use out of 14 days. Acceptability of tenofovir gel was assessed quantitatively and qualitatively. Women were invited to participate in qualitative focus groups within 6 weeks of completion of all other study activities. Male partners completed a quantitative follow-up acceptability assessment, and a qualitative individual semistructured interview following their partner's day 14 visit (R Rosen and A. Carballo-Dieguez, unpublished data).
The power of the study was characterized as follows: if the overall adverse event rate was expected to be 5%, 12 women in a given cohort would provide 88% power to exclude an adverse event rate > 35%. In addition, the upper bounds of the exact 95% confidence interval (CI) around the adverse event rates were 26.5% and 38.5% if the observed number of adverse events in a cohort of 12 women was 0 or 1, respectively. Because of the small sample size within each dose/frequency cohort, definitive comparisons among the cohorts could not be performed. The number and percentage of participants experiencing each type of adverse event was tabulated by severity and relationship to treatment for each cohort. Changes from baseline to follow-up within each cohort were assessed by a McNemar's test for dichotomous variables, or by a Wilcoxon signed rank test for continuous variables. All descriptive and inferential statistical analyses were performed by SCHARP (Statistical Center for HIV/AIDS Research and Prevention), using SAS (SAS Institute, Inc; Cary, North Carolina, USA) and StatXact (Cytel; Cambridge, Massachusetts, USA).
Eighty-four women were enrolled, including 48 HIV-negative, sexually abstinent women; 12 HIV-negative, sexually active women; 12 HIV-positive, sexually abstinent women; 12 HIV-positive sexually active women. There were 24 male HIV-seroconcordant partners. Female ethnicity was 45% African American; 35% Caucasian; 2% Asian; and 18% other; 19% women identified as Latina. Table 1 shows the dose and frequency of product application and Table 2 the schedule of clinical and laboratory evaluations.
There were no life-threatening adverse events or deaths reported in the study; 92% of the women reported at least one adverse event, with 87% of the participants having had at least one mild event. The most common adverse events were genital pruritus (23%), applicator site bruising (17%), applicator site erythema (17%), vaginal discharge (15%), irregular menses (13%), and metrorrhagia (11%). Specific adverse event patterns were not associated with gel concentration, sexual activity, or HIV status. Forty percent of the women classified at least one event as ‘moderate’, usually of limited duration. The majority, 70%, of the adverse events involved the ‘reproductive system and breast disorders’ (as per MedDRA coding) with the majority affecting the genital tract (Table 3); 32% of the participants reported gastrointestinal symptoms. The full cohort progression for the study was able to proceed because of the lack of a discernable adverse event pattern in relation to gel concentration or frequency of use. One severe adverse event occurred that was possibly product related, when a participant developed lower abdominal cramping necessitating an emergency room visit. The evaluating physician thought that pelvic inflammatory disease was the most likely diagnosis and prescribed empiric antibiotic therapy. There were three other severe adverse events, which were not judged to be product related: bronchitis, hyperglycemia, and suicidal ideation. One participant terminated product use after 10 days owing to a moderate adverse event (shallow vulvar ulcerations), which was probably product related.
At least one new pelvic or colposcopic examination finding developed during study participation in 48% of the women. Erythema was the most common finding. Three women had lesions that were possibly related to applicator or speculum trauma. Only one woman, with a history of eczema, had a severe colposcopic finding: a deep epithelial disruption that occurred 9 days after her last gel dose. However, the participant had skin ulcerations at the same time. Since it was possible (albeit unlikely) that her underlying problem was exacerbated by exposure to the gel, the event was classified as ‘possibly product related’. None of the participants experienced significant changes in any laboratory tests while they used the gel.
Pharmacokinetic evaluation was carried out for 25 women; 14 of these (56%) had low, but detectable serum tenofovir levels at one or more time points following either first dose (day 0) or following multiple dosing (day 13) (Fig. 1). The maximum systemic tenofovir concentrations ranged from 3.0 to 25.8 ng/ml, with no clear dose–concentration relationship; the median value was 3.4 ng/ml, just above the assay's limit of quantification of 3.0 ng/ml. One participant had a plasma tenofovir concentration of 25.8 ng/ml 2 h after her first dose on day 0. Her next level, 4 h after her first dose, was 10.9 ng/ml. All subsequent values were < 10 ng/ml. The participant was asymptomatic with a normal pelvic examination at the time that the highest tenofovir level was detected. (For comparison, the plasma concentration 24 h after the oral administration of a single 300 mg dose of tenofovir DF at steady state would be close to 50 ng/ml ). The median maximum systemic tenofovir concentration for all subjects (3.4 ng/ml) corresponded to approximately 1% of the maximum and 7% of the minimum blood concentrations, at steady-state, after 300 ng oral tenofovir DF dosing .
Plasma and cervicovaginal lavage samples were obtained at days 0, 7 and 14 from the HIV-positive women in the study. Specimens were available for 22 women and 13 had detectable HIV RNA (> 400 copies/ml) in the plasma at day 0; 12 of the women followed up at 14 days had detectable HIV RNA in the plasma. Cervicovaginal lavage HIV RNA was detectable for only 2 of 22 specimens on day 0, at 491 and 730 copies/ml. None of the participants had detectable cervicovaginal HIV RNA at day 14. Table 4 includes the results of genotypic testing of all specimens that contained sufficient quantities of HIV RNA to analyze amino acid substitutions associated with tenofovir resistance or hypersusceptibility. None of the samples contained the K65R, 69SS mutations. Low-level tenofovir resistance mutations (M41L, L210M, T215I/Y) were detected in plasma from three women at baseline who were using nucleoside reverse transcriptase medications at enrollment (Table 4), but these were not changed after tenofovir exposure. The M184V mutation associated with hypersusceptibility to tenofovir was detected in plasma samples from four women, and all but one of them had other mutations. The T69N mutation, which may contribute to tenofovir resistance, was detected in a plasma samples from another nucleoside analogue-experienced participant, who also had the M184V mutation. One of the two day 0 cervicovaginal lavage samples contained the T215I mutation, which was also detected in the day 14 plasma sample from the same woman. No new reverse transcriptase resistance mutations were detected after gel exposure.
Among 76 participants who had Gram stains of vaginal fluid tested at both enrollment and day 14, 30 women had asymptomatic bacterial vaginosis at enrollment (using Nugent's criteria). Bacterial vaginosis was not detected in 15 of these women (50%) after 14 days of tenofovir gel use. One of 46 women without bacterial vaginosis at enrollment developed bacterial vaginosis after 14 days of gel use. Two women (3%) developed asymptomatic vaginal candidiasis after product use.
Adherence and acceptability
Non-adherence was defined as 3 or more days of missed product use (out of 14). Three women terminated early from the study: one woman was lost to follow-up and two women refused further participation, citing the burden of the study visit schedule and procedures, but did not indicate that product was unacceptable. It was impossible to determine whether the one woman lost to follow-up was a consequence of an adverse event. Of the 81 women who completed the 2 week course, 76 (94%) were fully adherent to the protocol and 94% said that they would definitely or probably use the gel if it were available and they wanted protection from HIV transmission; 81% of the male partners also agreed that they would use the gel under similar circumstances. The gel was liked by 79% of the women and 76% of the men. Additional acceptability parameters are being analyzed (R Rosen and A. Carballo-Dieguez, unpublished data).
This is the first-phase I clinical trial to study an antiretroviral agent as a vaginal microbicide. In this cohort of low-risk, HIV-negative and HIV-positive women, tenofovir gel was well tolerated. The 1% concentration used twice daily was as well tolerated as 0.3% gel used only once a day. The adverse events reported in the trial mainly involved the genital tract and were generally transient and mild. They did not result in product discontinuation, non-adherence, or long-term morbidity. There were four severe adverse events (all of them resolved), with only one being possibly product related, abdominal cramping.
The prevalence of genital tract symptoms with tenofovir gel was not significantly greater than those noted in recent studies of other microbicide candidates [29–34]. Priestly et al.  noted that the majority of women asked to keep daily diaries about vaginal findings often noted mild localized symptoms in the absence of gel exposure. Future microbicide studies may need placebo, and possibly non-intervention, controls to assess fully the specific effects of microbicides, given that mucosal abnormalities are often detected when colposcopic examinations are performed, independent of microbicide use . Although half the women with asymptomatic bacterial vaginosis experienced normalization of their vaginal microflora after applying the gel, the variable natural history of bacterial vaginosis  would suggest that longer duration studies of tenofovir gel are needed to understand chronic local effects. Although mild adverse events were common, the majority of women and their male partners reported they would likely use the gel if it were available. In other microbicide studies [29–34], some of the abnormal findings, including ulcerations and abrasions, have been associated with the applicator use, as was the case in this study. Safer, effective microbicide delivery devices are needed.
Approximately half of the women had detectible plasma tenofovir levels at least once in the pharmacokinetics subgroup. Systemic absorption may mean that investigations of tenofovir gel use over longer periods of time will be needed to assess whether there is any cumulative toxicity, or whether tissue penetration enhances the gel's protective effects. Tenofovir is a nucleotide analogue that has been associated with rare cases of nephrotoxicity, as well as bone demineralization . However, oral dosing with tenofovir DF results in systemic exposure that is > 10-fold of the values seen in this study. Since several hundred thousand HIV-positive patients have tolerated daily tenofovir DF well for up to 5 years, significant toxicity from tenofovir gel exposure is unlikely. The finding of subinhibitory, low-level plasma concentrations of tenofovir also raises questions about the selection for HIV resistance in undiagnosed HIV-positive individuals and those who might be at risk for HIV seroconversion. None of the HIV-positive women who had detectible plasma or cervicovaginal HIV RNA developed mutations associated with tenofovir resistance, which was reassuring. Since only 13 women had HIV RNA detected in either the blood or cervicovaginal compartment, and tenofovir gel exposure lasted only 2 weeks, longer-term studies of HIV-positive women exposed to tenofovir gel will be needed to evaluate whether chronic use could select for resistance mutations. Given that tenofovir gel was well tolerated and acceptable in this cohort, a safety study of tenofovir gel in at-risk women is now underway in New York and Pune, India, which may further support the rationale for efficacy trials of this promising new topical microbicide.
We would like to acknowledge all the members of the study team as well as investigators and staff at the participating sites and supportive organizations. Special appreciation is owed to the following: Harlem Hospital Center (Judith Absalon, Maryse Joseph, Amarillis Lugo), Bronx Lebanon Medical Center (Almando Infante, Maria Teresa Timoney, Norris Allen), The Miriam Hospital and Fenway Health Center (Deborah Kutenplon, Hiroko Bojarski, Jeffrey Peipert, Laurie Boardman, Amy Carlson, Susan Cu-Uvin, Irma Rodriguez, Robbie Singal), University of Pennsylvania (Johnnita Prince, Cynthia Bayer, Nancy Tustin), Family Health International (Beth Barrows, Beverly Bell, Anne Coletti), Central Laboratory (Estelle Piwowar-Manning, Charlotte Gaydos, Lorna Rabe), Fred Hutchinson Cancer Center (Karen Patterson, Barbara Richardson), and National Institute of Allergy and Infectious Diseases (Roberta Black and Scharla Estep). We deeply appreciate the participants who volunteered for this study. The editorial comments of Howard Jaffe (Gilead), Judith Absalon, Ian McGowan, Thomas Fleming, and Thomas Coates, and the administrative assistance of Lola Wright, in the preparation of this manuscript are greatly appreciated.
Sponsorship: This study was supported by the HIV Prevention Trials Network (HPTN) and sponsored by the National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, National Institute on Drug Abuse, National Institute of Mental Health, and Office of AIDS Research, of the National Institutes of Health, US Department of Health and Human Services (U01–AI-48016, U01–AI-48040, U01–AI-48014, U01–AI-46702 and U01–AI-46745) and Gilead Sciences, Inc.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
microbicide; women; HIV prevention; clinical trial; antiretroviral